Haloaromatic and haloaliphatic contamination is a major environmental concern. Pollution often comes from municipal, industrial, agricultural or military sources, and it can enter the food-chain where it may bioaccumulate and pose significant health risks. Even if the sources of pollution are eliminated, the legacy of decades of accumulation of these environmental contaminants will persist.
The reduction of these contaminants in soils by mechanical or chemical technologies to levels approved by the US Environmental Protection Agency is prohibitively costly and very time consuming. The phytoremediation approach, on the other hand, using natural clones of plants is promising and has attracted interest due to its potentially lower cost, low maintenance and sustainability. Plants can also enrich the soil and co-act with microbes in the rhizosphere in the process of remediating the contaminated soil. Plants are suitable for sites where the plume of contamination in the soil is directly accessible for the roots or where the pollutant can be gradually drained and the "plump and treat" procedure applied using constructed plant beds.
The use of plants for removing or stabilizing contaminants in soil and water is an emerging technology that has had many successes. A typical wetland designed for municipal wastewater treatment includes one or more wetland plant species planted in a substrate of sand, soil, or gravel. Wastewater is applied at the upper end and flows slowly down a gentle gradient. The wastewater can be applied onto the surface then collected at the bottom. Reed-beds, for example, are used very frequently in these systems with high efficiency.
Much of what has been learned from the design of wetlands for nutrient removal can be applied to plant beds designed for halogenated organics abatement. The Poaceae (grass) species are prime targets for efforts to identify and improve plant varieties for halogenated organic waste treatment technologies in different environments because many species are dominant, monoculture-forming, perennial vascular plants, and they can thrive in polluted environment while producing a relatively large biomass.
Dehalogenating enzymes are uncommon in higher organisms in general and have not been reported for plants as yet. In bacteria, there are several different types of dehalogenases grouped according to reaction mechanisms (Neidleman & Geigert, 1986; Fetzner & Lingens, 1994) which are discussed briefly below.
1) Reductive dehalogenases replace the halogen atom of a haloorganic compound with hydrogen (C--X(C--H+X). This reaction has been described in several species of aerobic (Johnston et al. 1972) and anaerobic bacteria(DeWeerd et al. 1991). No reductive dehalogenases have been purified to date.
2) The dehydrohalogenase reaction proceeds by simultaneous removal of the halogen atom and the adjacent hydrogen on the neighboring carbon (H--C--C--X(C.dbd.C+HX). This reaction works with certain halogenated cyclohexane derivatives and halogenated straight-chain hydrocarbons (Neidleman & Geigert, 1986). Little is known about these enzymes of which none has been purified. Dehydrohalogenation is not always due to enzymatic reaction, these compounds may decompose spontaneously at a certain rate.
3) Hydrolytic dehalogenases (halidohydrolases) replace the halogen atom with a hydroxyl group (C--X+OH(C--OH+X; Goldman et al. 1968) The reaction may be either oxidative or hydrolytic (Scholten et al. 1991). These enzymes are able to attack haloacetates (Smith et al. 1990) and haloaromatic compounds including 4-chlorobenzoate (Elsner et al. 1991; Scholten et al. 1991). Genes encoding halidohydrolases have been cloned from several bacterial species (Elsner et al. 1991) and the nucleotide sequence of the structural genes encoding one enzyme has been reported (Schmitz et al. 1992).
4) Halohydrin epoxidase simultaneously removes the halogen atom and a hydrogen from an adjacent hydroxyl group on the neighboring carbon (HO--C--C--X(C--CO--epoxide+HX) and forms an epoxy bridge. The only known source of this enzyme is a soil bacterium, Flavobacterium sp. (Castro & Bartnicki, 1968). No activity by this class of this enzymes on haloaromatic compounds has been reported or would be expected, but dehalogenation of some polysubstituted halophenois may occur.
5) Oxidative displacement enzymes. Specifically,
a) peroxidases have been described whose action breaks the carbon-halogen bond by polymerization of free radical intermediates; PA1 b) oxygenases break the carbon-halogen bond by oxidative hydroxylation of the position adjacent to the carbon bearing the halogen atom (Markus et al. 1984).
Monooxygenases participate in halophenol degradation in several species (Uotila et al. 1992).
In bacteria, many aromatic compounds, including haloaromatics, are dissimilated either through protocatechuate or catechol (o-diphenol) intermediates which are then metabolized to 3-oxoadipate which is subsequently converted to acetyl-CoA and succinate. Toluene, mandelate, benzoate, phenol and some halogenated aromatics are usually dissimilated via the catechol branch pathway (Reineke & Knackmuss, 1984; Chen et al. 1989).
There are two different modes of enzymatic ring cleavage to detoxify halogenated aromatic compounds. The cleavage of the diphenol between the vicinal hydroxyl groups is known as the ortho pathway catalyzed by catechol 1,2-dioxygenase and protocatechuate 2,3-dioxygenase (intradiol dioxygenase). Cleavage of adjacent to the vicinal hydroxyl groups is known as the meta pathway catalyzed by catechol 2,3-dioxygenase and protocatechuate 4,5-dioxygenase (extradiol dioxygenase, Chen et al. 1984). The distribution of various pathways among bacteria is very complex with different pathways operating in species of the same genus (Chen et al. 1984 a&b, 1989).
Bacteria capable of degrading chlorinated aromatics break down these chemicals via a chloro-substituted catechol, which is always ortho-cleaved by a chlorocatechol 1,2-dioxygenase. These modified ortho-cleavage pathway enzymes have wider substrate specificities than ordinary ortho-cleavage pathway enzymes. Modified ortho-cleavage pathway genes from three bacteria have been extensively characterized.
Biotransformation of halogenated aliphatic and aromatic compounds using microbial or enzymatic biocatalysts provide a biotechnological approach to environmental protection. Yet, application of microbial systems in environmental protection technology is still limited, because of certain disadvantages, such as the requirement that the co-metabolism in the microbes have a toxic co-inducer and that the enzymes may have a narrow substrate range and high product selectivity. If the microbes inoculated into the soil are to come into contact with the chemical, they may have to move in the soil and their survival in the competitive microbial milieu is a concern.
Trichloroethene is a small molecule widely used as a solvent and a degreasing agent in industry (Vogel et al. 1987). TCE is volatile and is very difficult to dehalogenate chemically. TCE is a carcinogen and may have many other toxic effects on living organisms (Miller & Guengrich, 1982). Because of industrial waste disposal, TCE is prevalent in soil, groundwater and air. It has been placed on the national priority list of the US Environmental Agency, identifying it as one of the most common pollutants in industrial countries (Omenn 1987). Over the past years, biological degradation of TCE began to emerge as the promising approach to control environmental contamination (Travis & Doty, 1990).
Both anaerobic and aerobic bacteria can convert TCE into other chemicals. However, anaerobic bacteria convert TCE into vinyl chloride, an even more toxic agent (Beak & Jaffe, 1989; Freedman & Gossett, 1989). Thus anaerobic bacteria are not generally screened for the TCE degradation functions.
Fortunately, a number of aerobic bacteria have been identified possessing TCE degradation capability. These bacteria include methanotrops (Wackett & Gibson, 1988), an ammonia-oxidizer (Vannelli et al. 1990), a propare-oxidizer (Wackett et al. 1989), and recombinant E.coli strains (Winter et al. 1989, Zylstra et al. 1989). These aerobic bacteria contain a variety of oxygenases. In order to induce expression of these oxygenases in these bacteria, a variety of co-pollutants, such as toluene, phenol, methane, isoprene, propane and 2,4-dichlorophenoxyacetic acid, must be present (Oldenhuis et al. 1989; Wackett et al. 1989). TCE thus serves as a co-oxidative substrate for these oxygenases.
The co-pollutant requirement for TCE degradation potentially limits the use of these bacteria in the environment. In addition, it has been shown that toxic oxidation products generated during the co-oxidation process may result in rapid death of the bacteria (Wackett & Householder, 1989). Thus the current strategy of bacterial degradation of TCE, therefore, is thus somewhat problematic.
As for dehalogenating enzymes in higher organisms, the cytochrome P-450 monooxygenase enzymes are the only known examples and are capable of reductive dehalogenation of alkyl halocarbons under anaerobic conditions (Dawson 1988). Other oxygenases may also participate in dehalogenation reactions, but until now the only such enzymes (other than P-450) were the dehaloperoxidases from a worm (discovered by one of the inventors: Chen et al. 1996). Unfortunately, the worm dehaloperoxidase enzyme produces dehalogenated quinones as end products and which are still toxic. A certain degree of biotransformation of TCE by poplars has also since been reported in April, 1997 (Newman et al. 1997). TCE is converted by poplars to still-toxic derivatives such as trichloroethanol, trichloroacetic acid, and dichloroacetic acid. Traces of radiolabeled carbon dioxide was also produced by poplar tissue cultures when dosed with [.sup.14 C] TCE. indicating very inefficient but detectable (1%-2%) mineralization of TCE. Purification and characterization of the enzyme(s) have not been reported.
Therefore, there remains a need for a cost-effective and efficient way of remediating halogenated organic contaminants in the soil.